A current limitation of human stem cell derived cardiomyocytes as a disease model system or for cell replacement therapy has been the inability to produce well characterized populations of distinct cardiomyocyte lineages. Additionally, it is critical to understand how in vitro generated cardiomyocyte lineages relate to those which develop in vivo. The scientific aims of our parent UO1 are to investigate mechanisms underlying myocardial phenotypes of genomic variants identified in previous GWAS studies. Toward this goal, we are optimizing protocols for high throughput generation of human induced pluripotent stem cells (hiPSCs) from a genetic cohort, and for efficient differentiation of hiPSCs to cardiomyocytes of distinct lineages. Specifically, Aim 2 of Phase I of our parent UO1 is to develop efficient protocols for purifying different lineages of hiPSC-derived cardiomyocytes. This original aim was to obtain high efficiency of cardiomyocyte differentiation, and identify cell surface markers specific to ventricular and atrial lineages to be utilized for FACS purification of mixed cardiomyocyte populations derived from hiPSC. We have made substantial progress on this aim. Using a monolayer protocol with defined media conditions, we can obtain greater than 80% yield of cardiomyocytes. We have also identified cell surface markers that facilitate purification of ventricular cells. However, the advent of single cell technologies offers an opportunity for a deeper investigation into subtypes of cardiomyocytes generated in our in vitro system, for example, not just ventricular versus atrial, but right versus left chamber specific cells, or distinct subtypes of conduction system cells. By single cell characterization of subsets of myocytes generated using our established in vitro system, we hope to identify cell surface markers or signaling pathways by which to purify or generate well defined myocyte subtypes which can then be utilized to model specific disease affecting myocyte subtypes. We will also correlate single cell genetic signatures obtained from in vitro generated myocytes with those of single cells from human fetal hearts to ascertain how in vitro lineages relate to their in vivo counterparts. The overall goal of this supplement to our original UO1 is to perform single cell analysis on in vitro and in vivo derived cardiomyocytes to further refine in vitro protocols to obtain subtypes of cardiomyocytes and to relate in vitro myocytes to i vivo counterparts. Accordingly, our Specific Aims are: (1) To identify subtypes and lineage hierarchies of cardiomyocytes generated in vitro during a highly efficient monolayer protocol; (2) To compare gene expression profiles of myocyte subtypes generated in vitro to gene expression profiles of their in vivo counterparts, and to physically map genetically classified in vitro subtypes to their corresponding subtypes in human fetal heart.